Oligonucleotides having phosphorothioate linkages of high chiral purity

ABSTRACT

Sequence-specific phosphorothioate oligonucleotides comprising nucleoside units which are joined together by either substantially all Sp or substantially all Rp phosphorothioate intersugar linkages are provided. Such sequence-specific phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are prepared by enzymatic or chemical synthesis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/297,703, filed Aug. 29, 1994, now U.S. Pat. No. 5,506,212which is a continuation of U.S. patent application Ser. No. 07/777,007,filed Oct. 16, 1991, now abandoned. This application also is acontinuation-in-part of U.S. patent application Ser. No. 08/058,023,filed May 5, 1993 now U.S. Pat. No. 5,521,302, which is a divisionalapplication of U.S. patent application Ser. No. 07/777,670, filed Oct.15, 1991 (issued as U.S. Pat. No. 5,212,295, issue date May 18, 1993),which is a continuation-in-part of U.S. patent application Ser. No.07/777,007. Each of the above-mentioned applications is commonlyassigned with this application, and the entire disclosures of each areherein incorporated by reference.

FIELD OF THE INVENTION

This invention is directed to sequence-specific phosphorothioateoligonucleotides comprising nucleosides joined by intersugar linkages,and to their synthesis and use. More particularly, the intersugarlinkages linking the nucleosides of oligonucleotides of the presentinvention are substantially pure all Sp or all Rp chiralphosphorothioate linkages. Such oligonucleotides are prepared viachemical or enzymatic synthesis. They are especially well suited asdiagnostics, therapeutics and research reagents.

BACKGROUND OF THE INVENTION

Oligonucleotides are known to hybridize to single-stranded RNA orsingle-stranded DNA. Hybridization is the sequence-specific base pairhydrogen bonding of bases of the oligonucleotides to bases-of target RNAor DNA. Such base pairs are said to be complementary to one another.

In determining the extent of hybridization of an oligonucleotide to acomplementary nucleic acid, the relative ability of an oligonucleotideto bind to the complementary nucleic acid may be compared by determiningthe melting temperature of a particular hybridization complex. Themelting temperature (T_(m)), a characteristic physical property ofdouble helices, denotes the temperature (° C.) at which 50% helical(hybridized) versus coil (unhybridized) forms are present. T_(m) ismeasured by using the UV spectrum to determine the formation andbreakdown (melting) of the hybridization complex. Base stacking whichoccurs during hybridization, is accompanied by a reduction in UVabsorption (hypochromicity). Consequently, a reduction in UV absorptionindicates a higher T_(m). The higher the T_(m), the greater the strengthof the bonds between the strands.

Oligonucleotides can be used to effect enzymatic cleavage of a targetRNA by using the intracellular enzyme RNase H. The mechanism of suchRNase H cleavage requires that a 2'-deoxyribofuranosyl oligonucleotidehybridize to a target RNA. The resulting DNA-RNA duplex activates theRNase H enzyme and the activated enzyme cleaves the RNA strand. Cleavageof the RNA strand destroys the normal function of the target RNA.Phosphorothioate oligonucleotides operate via this type of mechanism.However, for a DNA oligonucleotide to be useful for cellular activationof RNase H, the oligonucleotide must be reasonably stable to nucleasesin order to survive in a cell for a time period sufficient for RNase Hactivation. For non-cellular uses, such as use of oligonucleotides asresearch reagents, such nuclease stability may not be necessary.

Several publications of Walder et al. describe the interaction of RNaseH and oligonucleotides. Of particular interest are: (1) Dagle et al.,Nucleic Acids Research 1990, 18, 4751; (2) Dagle et al., AntisenseResearch And Development 1991, 1, 11; (3) Eder et al., J. Biol. Chem.1991, 266, 6472; and (4) Dagle et al., Nucleic Acids Research 1991, 19,1805. According to these publications, DNA oligonucleotides having bothunmodified phosphodiester internucleoside linkages and modifiedphosphorothioate internucleoside linkages are substrates for cellularRNase H. Since they are substrates, they activate the cleavage of targetRNA by RNase H. However, the authors further note that in Xenopusembryos, both phosphodiester linkages and phosphorothioate linkages arealso subject to exonuclease degradation. Such nuclease degradation isdetrimental since it rapidly depletes the oligonucleotide available forRNase H activation.

As described in references (1), (2) and (4), to stabilizeoligonucleotides against nuclease degradation while still providing forRNase H activation, 2'-deoxy oligonucleotides having a short section ofphosphodiester linked nucleotides positioned between sections ofphosphoramidate, alkyl phosphonate or phosphotriester linkages wereconstructed. While the phosphoamidate-containing oligonucleotides werestabilized against exonucleases, in reference (4) the authors noted thateach phosphoramidate linkage resulted in a loss of 1.6° C. in themeasured T_(m) value of the phosphoramidate containing oligonucleotides.Such a decrease in the T_(m) value is indicative of a decrease inhybridization between the oligonucleotide and its target nucleic acidstrand.

Applications of oligonucleotides as diagnostics, research reagents, andtherapeutic agents require that the oligonucleotides be transportedacross cell membranes or taken up by cells, appropriately hybridize totarget RNA or DNA, and subsequently terminate or disrupt nucleic acidfunction. These critical functions depend partly on the initialstability of oligonucleotides towards nuclease degradation. Further,these functions depend on specificity of the oligonucleotide for atarget DNA or RNA molecule.

A serious deficiency of oligonucleotides for these purposes is theirsusceptibility to enzymatic degradation by a variety of ubiquitousnucleases which may be intracellularly and extracellularly located.Unmodified, "wild type", oligonucleotides are not useful as therapeuticagents because they are rapidly degraded by nucleases. Therefore,modification of oligonucleotides for conferring nuclease resistance onthem has been the primary focus of research directed towards thedevelopment of oligonucleotide therapeutics and diagnostics.

Modifications of oligonucleotides to enhance nuclease resistance hasgenerally taken place on the sugar-phosphate backbone, particularly onthe phosphorous atom. Phosphorothioates have been reported to exhibitresistance to nucleases. In addition, phosphorothioate oligonucleotidesare generally more chemically stable than natural phosphodiesteroligonucleotides. Phosphorothioate oligonucleotides also exhibitsolubility in aqueous media. Further, phosphorothioateoligonucleotide-RNA heteroduplexes can serve as substrates forendogenous RNase H. Additionally, phosphorothioate oligonucleotidesexhibit high thermodynamic stability. However, while the ability of anoligonucleotide to bind to a target DNA or RNA with fidelity is criticalfor its hybridization to the target DNA or RNA, modifications at thephosphorous atom of the oligonucleotides, while exhibiting variousdegrees of nuclease resistance, have generally suffered from inferiorhybridization properties [Cohen, J. S., Ed., Oligonucleotides: AntisenseInhibitors of Gene Expression (CRC Press, Inc., Boca Raton, Fla., 1989].

One reason for this inferior hybridization may be the prochiral natureof the phosphorous atom. Modifications on the internal phosphorous atomof modified phosphorous oligonucleotides results in Rp and Spstereoisomers. Modified phosphorus oligonucleotides obtained thus far,wherein the resulting molecule has nonsymmetrical substituents, havebeen racemic mixtures having 2^(n) isomers, with n equal to the numberof phosphorothioate intersugar linkages in the oligonucleotide. Thus, a15-mer phosphorothioate oligonucleotide, containing 14 asymmetriccenters has 2¹⁴ or 16,384 diastereomers. In view of this, in a racemicmixture, only a small percentage of the oligonucleotides are likely tospecifically hybridize to a target mRNA or DNA with sufficient affinity.

Chemically synthesized phosphorothioate oligonucleotides having chirallypure intersugar linkages had thus far been limited to molecules havingonly one or two diastereomeric intersugar linkages. Until recently, theeffects of induced chirality in chemically synthesized racemic mixturesof sequence-specific phosphorothioate oligonucleotides had not beenassessed since synthesis of oligonucleotides having chirally pureintersugar linkages had yet to be accomplished by automated synthesis.This was due to the non-stereospecific incorporation of sulfur duringautomated synthesis. For example, Stec et al., J. Chromatography,326:263 (1985), synthesized certain oligonucleotide phosphorothioateshaving racemic intersugar linkages, however, they were able to resolveonly the diastereomers of certain small oligomers having one or, atmost, two diastereomeric phosphorous intersugar linkages.

However, Stec et al. [Nucleic Acids Res., 19:5883 (1991)] subsequentlyreported the automated stereo-controlled synthesis of oligonucleotides.The procedure described in the above-mentioned reference utilizesbase-catalyzed nucleophilic substitution at a pentavalentphosphorothioyl center.

The synthesis of phosphorothioates having all Rp intersugar linkagesusing enzymatic methods has been investigated by several authors[Burgers and Eckstein, J. Biological Chemistry, 254: 6889 (1979); Guptaet al., J. Biol. Chem., 256: 7689 (1982); Brody and Frey, Biochemistry,20: 1245 (1981); and Eckstein and Jovin, Biochemistry, 2:4546 (1983)].Brody et al. [Biochemistry, 21:2570 (1982)] and Romaniuk and Eckstein,[J. Biol. Chem., 257:7684 (1982)] enzymatically synthesized poly TpA andpoly ApT phosphorothioates, while Burgers and Eckstein [Proc. Natl.Acad. Sci. U.S.A., 75:4798 (1978)] enzymatically synthesized poly UpAphosphorothioates. Cruse et al. [J. Mol. Biol., 192:891 (1986)] linkedthree diastereomeric Rp GpC phosphorothioate dimers via naturalphosphodiester bonds into a hexamer.

The relative ability of an oligonucleotide to bind to complementarynucleic acids may be compared by determining the melting temperature ofa particular hybridization complex. The melting temperature (T_(m)), acharacteristic physical property of double helixes, denotes thetemperature (° C.) at which 50% helical versus coil (unhybridized) formsare present. T_(m) is measured by using the UV spectrum to determine theformation and breakdown (melting) of hybridization. Base stacking whichoccurs during hybridization, is accompanied by a reduction in UVabsorption (hypochromicity). Consequently a reduction in UV absorptionindicates a higher T_(m). The higher the T_(m), the greater the strengthof the binding of the strands. Non-Watson-Crick base pairing has astrong destabilizing effect on the T_(m).

In a preliminary report [Stec, J. W., Oligonucleotides as AntisenseInhibitors of Gene Expression: Therapeutic Implications, Meetingabstracts, Jun. 18-21, 1989], thymidine homopolymer octamers having allbut one linkage being modified phosphate linkages ("all except one") Rpstereoconfiguration or "all except one" Sp stereoconfiguration in theintersugar linkages were formed from two thymidine methylphosphonatetetrameric diastereomers linked by a natural phosphodiester bond. It wasnoted that a Rp "all except one" methylphosphonate non-sequence-specificthymidine homooctamer, i.e. (dT)₈ having all but one Rp intersugarlinkage, formed a thermodynamically more stable hybrid (Tm 38° C.) witha 15-mer deoxyadenosine homopolymer, i.e. (dA)₁₅, than a hybrid formedby a similar thymidine homopolymer having "all except one" Spconfiguration methylphosphonate linkages and of d(A)₁₅ (Tm<0° C.), i.e.a d(T)₁₅ having all but one Sp intersugar linkage. A hybrid between(dT)₈ having natural phosphodiester linkages, i.e. octathymidylic acid,and d(A)₁₅ was reported to have a Tm of 14° C.

More recently, Ueda et al. [Nucleic Acids Research, 19:547 (1991)]enzymatically synthesized mRNAs intermittently incorporating Rpdiastereomeric phosphorothioate linkages for use in translation systems.Ueda et al. employed T7 coliphane DNA having seventeen promoters and onetermination site for T7 RNA polymerase. In vitro synthesis by T7 RNApolymerase produced mRNAs having from several hundred to tens ofthousands of nucleotides.

Backbone chirality may also affect the susceptibility of aphosphorothioate oligonucleotide-RNA heteroduplex to RNase H activity.The ability to serve as a template for RNAse H has significanttherapeutic implications since it has been suggested that RNAse H causescleavage of the RNA component in an RNA-DNA oligonucleotideheteroduplex. With oligonucleotides containing racemic mixtures of Rpand Sp intersugar linkages, it is not known if all phosphorothioateoligonucleotides can function equally as substrates for RNase H. For avariety of catalytic reactions, hydrolysis of the phosphodiesterbackbone of nucleic acids proceeds by a stereospecific mechanism (anin-line mechanism) and inversion of configuration. Therefore, there maybe only a small percentage of oligonucleotides in a racemic mixture thatcontain the correct chirality for maximum hybridization efficiency andtermination of translation. Thus, increasing the percentage ofphosphorothioate oligonucleotides that can serve as substrates for RNAseH in a heteroduplex will likely lead to a more efficacious compound forantisense therapy.

To enhance hybridization fidelty, phosphorothioate oligonucleotideshaving substantially chirally pure intersugar linkages are greatlydesired. Further, such phosphorothioate oligonucleotides havingsubstantially chirally pure intersugar linkages would lead to moreefficacious therapeutic compounds. However, until now little success hasbeen achieved in synthesizing such molecules. Therefore, simple methodsof synthesizing phosphorothioate oligonucleotides having substantiallychirally pure intersugar linkages are greatly desired.

It has been recognized that nuclease resistance of oligonucleotides andfidelity of hybridization are of great importance in the development ofoligonucleotide therapeutics. Oligonucleotides possessing nucleaseresistance are also desired as research reagents and diagnostic agents.

OBJECTS OF THE INVENTION

It is an object of this invention to provide sequence-specificphosphorothioate oligonucleotides having substantially chirally pure,either all Rp or all Sp, intersugar linkages.

It is a further object of this invention to provide phosphorothioateoligonucleotides having all Rp or all Sp intersugar linkages that arespecifically hybridizable to target DNA or RNA.

It is another object of this invention to provide methods for synthesisof sequence-specific phosphorothioate oligonucleotides havingsubstantially chitally pure intersugar linkages.

These and other objects of the present invention shall become apparentto persons skilled in the art to which this invention pertains giventhis specification and the claims appended hereto.

SUMMARY OF THE INVENTION

In accordance with this invention, phosphorothioate oligonucleotideshaving all nucleoside units joined together by either substantially allSp phosphorothioate intersugar linkages or substantially all Rpphosphorothioate intersugar linkages are provided. Preferably, thephosphorothioate oligonucleotides of the present invention arecomplementary to at least a portion of the sequence of a target RNA orDNA.

Further, in accordance with the present invention, chemical andenzymatic methods of synthesizing sequence-specific phosphorothioateoligonucleotides having substantially chirally pure intersugar linkagesare provided wherein said phosphorothioate oligonucleotides arecomprised of at least 10 nucleoside units joined together by eithersubstantially all Rp or substantially all Sp intersugar linkages.Preferably, the phosphorothioate oligonucleotides are comprised of about10 to about 50 nucleoside units joined by substantially chirally pureintersugar linkages. More preferably, said phosphorothioateoligonucleotides are comprised of about 15 to about 30 nucleoside unitsjoined by substantially chirally pure intersugar linkages. Mostpreferably, said phosphorothioate oligonucleotides are comprised ofabout 17 to 21 nucleoside units joined together by substantiallychirally pure intersugar linkages. Said methods comprise combiningsequence primers, templates, and an excess of all four chirally purenucleoside 5'-O-(1-thiotriphosphates). Said methods further includesynthesizing complementary oligonucleotides by the addition ofpolymerase followed by cleavage of the primer from the complementaryoligonucleotides. In addition, said methods are comprised ofdisassociating said complementary oligonucleotides from said template.

In alternative embodiments of the present invention methods ofsynthesizing sequence-specific phosphorothioate oligonucleotides havingsubstantially chirally pure intersugar linkages, sequence primers,templates and racemic mixtures of nucleoside 5'-O-(1-thiotriphosphates)are combined. Phosphorothioate oligonucleotides having substantiallychirally pure intersugar linkages and which are complementary to thetemplate are synthesized by the addition of polymerase and a selectedmetal ion. Oligonucleotides thus synthesized are dissociated from thetemplate and primer.

Phosphorothioate oligonucleotides having substantially chirally pureintersugar linkages are useful for increasing the thermodynamicstability of heteroduplexes formed with target RNA and DNA and to elicitRNase H activity. Further, oligonucleotides of the present invention areuseful for modulating the activity of RNA.

DETAILED DESCRIPTION OF THE INVENTION

The phosphorous atom in a phosphodiester linkage of an oligonucleotidecan be described as being "pro-chiral." Once a non-bonding oxygen atomof the phosphodiester linkage is replaced or modified, a chiralsugar-phosphate linkage is generated. The resulting intersugar linkageis either an Sp intersugar linkage or an Rp intersugar linkage.Replacement of a non-bonding oxygen atom of the natural phosphodiesterlinkage with sulfur to obtain a phosphorothioate linkage results in thegeneration of a chiral center and affords Sp and Rp diastereomers.Molecules wherein substantially all of the phosphorous atoms in thesugar backbone are either Sp or Rp are referred to herein as chirallypure.

Ribonucleoside- (NTPαS) and2'-deoxyribonucleoside-5'-O-(1-thiotriphosphates) (dNTPαS) have beensynthesized as Sp and Rp racemic mixtures using the methodology ofLudwig and Eckstein [J. Org. Chem., 631 (1989)]. In this exemplarysynthetic scheme, unprotected nucleosides can be reacted with2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, which phosphitylates the5'-hydroxyl group. Subsequent reaction with pyrophosphate yields cyclictriphosphate derivatives which are reactive to sulfur, yielding mixturesof Rp and Sp nucleoside 5'-O-(1-thiotriphosphates), i.e.α-thiotriphosphates. The products can be purified by DEAE-Sephadexchromatography and identified by NMR spectroscopy (by characteristic Rpor Sp chemical shifts).

As is shown in the examples below, pure Rp and Spnucleoside-5'-O-(1-thiotriphosphates) diastereomers can be readilyisolated on a preparative scale using, for example, reverse phase HPLCchromatography. Such HPLC-isolated nucleotide diastereomers can befurther characterized by analytical HPLC comparisons with commercialsamples of such Rp and Sp nucleoside 5'-O-(1-thiotriphosphates)diastereomers.

Enzymatic synthesis of sequence-specific natural oligonucleotides, i.e.natural phosphodiester oligonucleotides, can be effected by the use ofan appropriate nuclease in the presence of a template and primer. In alike manner, racemic mixtures of phosphorothioate oligonucleotideshaving chirally mixed intersugar linkages can be synthesized. Accordingto the present invention, such enzymatic synthesis can also be expandedto include the synthesis of sequence specific phosphorothioateoligonucleotides having substantially chirally pure intersugar linkagesby utilizing enantiomerically pure all-Sp or all-Rp nucleoside5'-O-(1-thiotriphosphates) as substrates for appropriate nucleases inthe presence of a sequence-specific template and a primer. For example,commercially available DNA polymerase Sequenase™ (U.S. Biochemical,Inc., Cleveland, Ohio) may be used to synthesize phosphorothioateoligonucleotides using a phosphodiester oligonucleotide template and aracemic phosphorothioate oligonucleotide primer. Using this polymeraseboth phosphodiester and phosphorothioate primers may be extended.

Yields of enzymatically synthesized phosphorothioate oligonucleotidescan be optimized by repetitive additions of template and primer, byrepetitive additions of polymerase, by repetitive additions ofnucleoside triphosphates or by combinations of some or all of these. Forinstance, repetitive additions of template and primer results inmaximizing yields via an enzymatic cascade. Further optimization can beachieved by pre-hybridization of template and primer together in systembuffer, followed by cooling and addition of nucleoside triphosphates andpolymerase.

A suitable polymerase may be selected to yield either DNA or RNAphosphorothioate oligonucleotides. Such polymerases include but are notnecessarily limited to T7 DNA polymerase, modified T7 DNA polymerasessuch as the above referenced Sequenase™, E. coli DNA polymerase, DNApoly Klenow fragment polymerase, M. luteus polymerase, T4 bacteriophagepolymerase, modified T4 DNA polymerase, T7 RNA polymerase and E. coliRNA polymerase.

The enzymatic synthesis proceeds with inversion of configuration aboutthe chiral center of the phosphorous atom. Thus, use of all Spα-thiotriphosphates yields substantially all Rp phosphorothioateoligonucleotides while use of all Rp α-thiotriphosphates yieldssubstantially all Sp phosphorothioate oligonucleotides. In an alternateembodiment of the invention, phosphorothioate oligonucleotides may besynthesized from racemic mixtures ofnucleoside-5'-O-(1-thiotriphosphates) utilizing metal ions in reactionsolutions to promote preferential incorporation of one or the other ofthe chiral α-thiotriphosphates. As noted above, polymerase synthesis ofphosphorothioate oligonucleotides is accomplished with inversion ofconfiguration about the chiral center of the precursornucleoside-α-thiotriphosphate. While not wishing to be bound by theory,it is believed that optimization of an all Rp configuration may beaccomplished by addition of a high concentration of magnesium ion in thereaction buffer utilizing, for instance, an E. coli polymerase. In alike manner, again while not wishing to be bound by theory, an all Spconfiguration might be obtained by utilizing a high manganese ionconcentration in the reaction buffer.

In accordance with the present invention, "substantially all" is meantto include all oligonucleotides in which at least 75% of the intersugarlinkages are chirally pure. More preferably, oligonucleotides havingfrom about 85% to about 100% chirally pure intersugar linkages aresubstantially chirally pure. Most preferably, oligonucleotides havingfrom about 95% to about 100% chirally pure intersugar linkages aresubstantially chirally pure.

In the context of this invention, the term "phosphorothioateoligonucleotide" includes phosphorothioate oligonucleotides formed fromnaturally occuring bases, sugars and phosphorothioate linkages.Naturally occuring bases include adenine, guanine, cytosine, thymine anduracil. Natural sugars include β-D-ribofuranosyl andβ-D-2'-deoxyerythro-pentofuranosyl. To the extent thatnucleoside-5'-O-(1-thiotriphosphate) analogs are substrates for suitablepolymerases, "phosphorothioateoligonucleotides" also include modifiedbases or modified sugars incorporated within the phosphorothioatenucleotide units of the oligonucleotides. Modified bases of theoligonucleotides of this invention include adenine, guanine, adenine,cytosine, uracil, thymine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halocytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil,4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine,8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substitutedadenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine,8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substitutedguanines, other aza and deaza uracils, other aza and deaza thymidines,other aza and deaza cytosines, other aza and deaza adenines, other azaand deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.The sugar moiety may be deoxyribose or ribose. The oligonucleotides ofthe invention may also comprise modified nucleobases or nucleobaseshaving other modifications consistent with the spirit of this invention,and in particular modifications that increase their nuclease resistancein order to facilitate their use as therapeutic, diagnostic or researchreagents.

Oligonucleotides of the invention can be utilized as diagnostics,therapeutics and as research reagents. They can be utilized inpharmaceutical compositions by adding an effective amount of anoligonucleotide of the invention to a suitable pharmaceuticallyacceptable diluent or carrier. They further can be used for treatingorganisms having a disease characterized by the undesired production ofa protein. The organism can be contacted with an oligonucleotide of theinvention having a sequence that is capable of specifically hybridizingwith a strand of target nucleic acid that codes for the undesirableprotein.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.In general, for therapeutics, a patient in need of such therapy isadministered an oligonucleotide in accordance with the invention,commonly in a pharmaceutically acceptable carrier, in doses ranging from0.01 μg to 100 g per kg of body weight depending on the age of thepatient and the severity of the disease state being treated. Further,the treatment regimen may last for a period of time which will varydepending upon the nature of the particular disease, its severity andthe overall condition of the patient, and may extend from once daily toonce every several years. Following treatment, the patient is monitoredfor changes in his/her condition and for alleviation of the symptoms ofthe disease state. The dosage of the oligonucleotide may either beincreased in the event the patient does not respond significantly tocurrent dosage levels, or the dose may be decreased if an alleviation ofthe symptoms of the disease state is observed, or if the disease statehas been ablated.

In some cases it may be more effective to treat a patient with anoligonucleotide of the invention in conjunction with other traditionaltherapeutic modalities.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual oligonucleotides, andcan generally be estimated based on EC₅₀ s found to be effective in invitro and in vivo animal models. In general, dosage is from 0.01 μg to100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every several years.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the oligonucleotide is administered in maintenance doses,ranging from 0.01 μg to 100 g per kg of body weight, once or more daily,to once every several years.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, or intrathecal or intraventricularadministration.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

Compositions for intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives.

Phosphorothioate oligonucleotides of the present invention can becontrasted with both natural phosphodiester oligonucleotides and racemicphosphorothioate nucleotides as to their effects on hybridization,nuclease resistance and RNAse H activity. In like manner, purephosphorothioate oligonucleotides having substantially chirally pureintersugar linkages may also be assessed for their ability to increaseeffectiveness of therapy in in vivo test systems. Such increase ineffectiveness of therapy might include attributes such aspharmacokinetics or metabolism, toxicology, disposition (i.e. absorptionand distribution), and species comparisons.

Homopolymers having all Rp or all Sp intersugar linkages have beenuseful for initial studies of stability and other characteristics.However, these oligonucleotides have little use therapeutically as theyare not specific for target molecules. Phosphorothioate oligonucleotideshaving specific sequences are necessary in order to specificallyhybridize to target nucleic acids.

Sequence-specificphosphorothioateoligonucleotides having substantiallychirally pure intersugar linkages are useful to increase thethermodynamic stability of heteroduplexes with target RNA and DNA and toelicit RNase H activity.

Radiolabeling can be used to assist in the identification ofphosphorothioate oligonucleotides having substantially chirally pureintersugar linkages. For racemic phosphorothioate oligonucleotidessynthesized on an automated synthesizer, [³⁵ S] (radiolabeled elementalsulfur) can be used for oxidation of the hydrogen-phosphonate oligomersobtained from the synthesizer. Labeling of enzymatically synthesizedphosphorothioate oligonucleotides can be accomplished with [α-³² P]ATPand ligase or [α-³⁵ S]ATPs in the polymerase reaction. Also,radiolabeled nucleoside triphosphates can be used in probe andsequencing analysis. Autoradiograms are prepared in standard manners.

Templates of the present invention are most preferably areas of nucleicacid sequence which direct synthesis of disease-potentiating proteins.Short oligonucleotides that base pair to a region of said templateoligonucleotide act as primers which form the starting point foroligonucleotide synthesis by polymerases.

Phosphorothioate oligonucleotides having substantially chirally pureintersugar linkages may be synthesized using a primer which may beselected to have a site thereon that is susceptible to nucleasecleavage, for example, restriction endonuclease cleavage. Said cleavagesite may be located at the 3' end of said primer. Cleavage at said siteby an appropriate restriction endonuclease results in oligonucleotidesderiving a first 5' end nucleoside from said primer. Additionalnucleosides of said phosphorothioate oligonucleotides of the presentinvention are those nucleoside chiral thiotriphosphates added viaenzymatic means.

By selecting appropriate restriction nucleases in conjunction withselected primers, various 5'-terminal nucleosides of desiredphosphorothioate oligonucleotides are appropriately positioned at the 5'end of a phosphorothioate nucleotide. Thus, any endonuclease recognitionsite can be designed as long as the staggered cut results in onenucleoside from the primer being the first 5' nucleoside of the newlysynthesized sequence specific phosphorothioate oligonucleotide of theinvention. This results in the generation of different nucleosides on 5'ends of enzymatically synthesized phosphorothioate oligonucleotides ofthe invention.

Upon completion of enzymatic extension of said primer on an appropriatetemplate of a desired sequence, phosphorothioate oligonucleotides of theinvention may be released from said primer by use of appropriatenuclease. For example, for incorporation of a guanosine nucleoside atthe 5' end of desired phosphorothioate oligonucleotides, a primer havingan CTGCAG sequence at its 3' terminal end may be used. Use of a Pst 1restriction nuclease then may cleave the A-G linkage. The guanosinenucleoside component of this A-G linkage may thus incorporated as a 5'terminal nucleoside of desired phosphorothioate oligonucleotides. Otherrestriction endonuclease include but are not limited to BamH1, Smal andHind III restriction endonucleases.

Oligonucleotides still associated with said template may be dissociatedfrom said template and then purified by gel electrophoresis and/orchromatography. For example, suitable purification can be accomplishedutilizing standard polyacrylamide/urea gel electrophoresis coupled withSepPac (Millipore, Miford, Mass.) chromatography. Another usefulchromatographic technique that may be employed is HPLC chromatography.

Chiral phosphorothioate oligonucleotides of the present invention mayalso be chemically synthesized via 1,3,2 -oxathiaphospholaneintermediates as described by Stec et al. [Nucleic Acids Res., 19: 5883(1991) ] and Stec and Lesnikowski [Methods in Molecular Biology, S.Agrawal, Ed., Volume 20, p. 285, 1993].

Phosphorothioate oligonucleotides having substantially chirally pureintersugar linkages which are synthesized according to methods of thepresent invention may be analyzed by a number of methods. For example,configuration analysis of resulting sequence- specific phosphorothioateoligonucleotides having subtantially chirally pure all Sp or all Rpintersugar linkages may be determined by the use of [³¹ P] NMR chemicalshifts. Such chemical shifts have been used to identify the Rp epimer ofa phosphorothioate dinucleotide [Ludwig and Eckstein, J. Org. Chem.,631-635 (1989)].

The fidelity of sequences of phosphorothioate oligonucleotides of theinvention can be determined using the sensitivities of heteroduplexes toS1 nuclease.

The sequence of the phosphorothioate oligonucleotides can be furthersubstantiated by labeling the 3' hydroxyl groups of phosphorothioateoligonucleotides with [alpha-³² P] cordycepin triphosphate, i.e.3'-deoxyadenosine-5'-triphosphate. The resultant oligonucleotides may besubjected to enzymatic degradation.

The relative ability of phosphorothioate oligonucleotides havingsubstantially chirally pure intersugar linkages to bind to complementarystrands is compared by determining the melting temperature of ahybridization complex of a phosphorothioate oligonucleotide havingsubstantially chirally pure intersugar linkages and its complementarystrand. The melting temperature (T_(m)), a characteristic physicalproperty of double helixes, denotes the temperature in degreescentigrade at which 50% helical versus coiled (unhybridized) forms arepresent. T_(m) is measured by using the UV spectrum to determine theformation and breakdown (melting) of hybridization. Base stacking, whichoccurs during hybridization, is accompanied by a reduction in UVabsorption (hypochromicity). Consequently a reduction in UV absorptionindicates a higher T_(m). The higher the T_(m), the greater the strengthof the binding of the strands. Non Watson-Crick base pairing has astrong destabilizing effect on the T_(m). Consequently, as close tooptimal fidelity of base pairing as possible is desired to have optimalbinding of an oligonucleotide to its targeted RNA.

Phosphorothioate oligonucleotides of the invention can also be evaluatedfor their resistance to the degradative ability of a variety ofexonucleases and endonucleases. Phosphorothioate oligonucleotides may betreated with nucleases and then analyzed, as for instance, bypolyacrylamide gel electrophoresis (PAGE) followed by staining with asuitable stain such as Stains All™ (Sigma Chem. Co., St. Louis, Mo.).Degradation products may be quantitated using laser densitometry.

Fetal calf and human serum may be used to evaluate nucleolytic activityon phosphorothioate oligonucleotides having substantially chirally pureintersugar linkages. For instance, a phosphorothioate oligonucleotidehaving substantially all Rp intersugar linkages may be evaluated in thismanner. Testing on combinations of 3' or 5' end capped (having one orseveral phosphorothioate linkages per cap) molecules may be used toestablish a combination that yields greatest nuclease stability. Cappingcan be effected by chemically synthesizing the cap portion of a sequenceusing purified Rp monomers followed by incorporation of said cap intooligonucleotides on the DNA synthesizer. Analysis involving capping candetermine the importance of chirality on nucleolytic stability and thenumber of linkages required to obtain maximum stability.

The sensitivity of phosphorothioate oligonucleotide-RNA heteroduplexesto the catalytic activity of RNase H can also be assessed. Aphosphorothioate oligonucleotide can be incubated with a radiolabeledtarget mRNA (synthesized as for instance via T7 RNA polymerase) atvarious temperatures for hybridization. Heteroduplexes can then beincubated at 37° C. with RNase H from E. coli according to the procedureof Minshull and Hunt [Nuc. Acid Res., 6433 (1986)]. Products may then beassessed for RNase H activity by Northern Blot analysis wherein productsare electrophoresed on a 1.2% agarose/formaldehyde gel and transferredto nitrocellulose. Filters may then be probed using a random primer [³²P]-labeled cDNA complementary to target mRNA and quantitated byautoradiography. Comparisons between different phosphorothioate analogscan be made to determine the impact of chirality on the ability to actas a substrate for RNase H when complexed to RNA.

Comparisons of the susceptibility of heteroduplexes to the catalyticaction of E. coli RNase H and mammalian RNAse H can be performed.Heteroduplexes can be incubated in rabbit reticulocyte lysates underconditions of translation and assayed via Northern blot analysis forcatalytic cleavage of mRNA by endogenous RNase H. This allows fordetermination of the effects of chirality on mammalian RNAse H activity.

Phosphorothioate oligonucleotides having substantially chirally pureintersugar linkages can also be evaluated for inhibition of geneexpression in cell culture model systems. To determine if aphosphorothioate oligonucleotide having substantially pure chirally pureintersugar linkages is more potent or a more specific inhibitor of geneexpression, a phosphorothioate oligonucleotide having substantiallychirally pure intersugar linkages designed to target reporter genes maybe synthesized and tested in cell culture models of gene expression. Theuse of the vector pSV2CAT has previously been described to measureantisense effects on gene expression [Henthorn et al., Proc. Natl. Acad.Sci. U.S.A., 85:6342 (1988)]. This vector contains the bacterialchloramphenicol acetyl transferase gene under regulatory controls of theSV40 promoter. Utilizing a 15-mer phosphorothioate oligonucleotidehaving all Rp intersugar linkages of a sequence complementary to theinitiation of translation of the CAT mRNA, pSV2CAT may be transfectedinto HeLa cells and, following treatment of the cells for 48 hr with aphosphorothioate oligonucleotide having all Rp intersugar linkages, CATactivity may then be assayed in the cells. The activity of aphosphorothioate having substantially chirally pure intersugar linkagesin inhibition of gene expression may then be compared directly with achemically synthesized random phosphorothioate having diastereomericintersugar linkages and natural phosphodiester oligonucleotides of thesame sequence.

The vector pSV2APAP [Marcus-Sekura et al., Nucleic Acids Research,15:5749 (1987)] contains the mammalian placental alkaline phosphatasegene (PAP). This can also be used as a reporter for measuring antisenseeffects on gene expression. PAP has advantages over CAT as a reportergene in that it is a mammalian gene, rather than a bacterial gene thatcontains introns and other RNA processing signals. It is presentlybelieved that PAP expression mimics more closely the events in naturalmammalian gene expression. A 15-mer phosphorothioate oligonucleotidehaving substantially chirally pure intersugar linkages as describedabove for the CAT mRNA can be examined in parallel with chemicallysynthesized racemic phosphorothioate and natural phosphodiesteroligonucleotides having similar sequences. The PAP and CAT reporterconstructs are used as controls in reciprocal experiments to test fornon-specific effects on gene expression.

Additionally, phosphorothioate oligonucleotides having substantiallychirally pure intersugar linkages can be evaluated as to their abilityto act as inhibitors of RNA translation in vivo. Various therapeuticareas can be targeted for such manipulation by oligonuclotides of thepresent invention. One therapeutic area is hepatitis caused by HepatitisC virus (HCV). The following phosphorothioate oligonucleotides haveapplication in the treatment of HCV hepatitis: Oligo #259,CCTTTCGCGACCCAACACTA (SEQ ID NO:1), Oligo #260, GCCTTTCGCGACCCAACACT(SEQ ID NO:2), Oligo #270, GTACCACAAGGCCTTTCGCG (SEQ ID NO:3), Oligo#330, GTGCTCATGGTGCACGGTCT (SEQ ID NO:4) and Oligo #340,TTTAGGATTCGTGCTCATGG (SEQ ID NO:5). Another therapeutic areainflammatory diseases mediated by intercellular cell adhesion molecule(ICAM-1). Oligonucleotides having application in the treatment ofinflammatory diseases include: ISIS-2302, GCCCAAGCTGGCATCCGTCA (SEQ IDNO:6). Another therapeutic area includes infections caused bycytomegalovirus (CMV). ISIS-2922 is a phosphorothioate oligonucleotidehaving application in the treatment of CMV retinitis, and has thesequence GCGTTTGCTCTTCTTCTTGCG (SEQ ID NO:7). Another therapeutic areaincludes PKC-α-mediated cancers. ISIS-3521 is a phosphorothioateoligonucleotide having application in the treatment of such cancers, andhas the sequence GTTCTCGCTGGTGAGTTTCA (SEQ ID NO:8). A furthertherapeutic area includes C-tar kinase-mediated cancers. ISIS-5132 is aphosphorothioate oligonucleotide having application in the treatment ofsuch cancers, and has the sequence TCCCGCCTGTGACATGCATT (SEQ ID NO:9). Astill further therapeutic area includes cancers mediated by Ha-ras orKi-ras. The following phosphorothioate oligonucleotides have applicationin the treatment of such cancers: ISIS-2503, TCCGTCATCGCTCCTCAGGG (SEQID NO:10), ISIS-2570, CCACACCGACGGCGCCC (SEQ ID NO:11), and ISIS-6957,CAGTGCCTGCGCCGCGCTCG (SEQ ID NO:12). In the above sequences, individualnucleotide units of the oligonucleotides are listed in a 5' to 3'direction from left to right.

In the context of this invention, "hybridization" shall mean hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleotide units. For example,adenine and thymine are complementary nucleobases which pair through theformation of hydrogen bonds. "Complementary," as used herein, alsorefers to sequence complementarity between two nucleotide units. Forexample, if a nucleotide unit at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide unit atthe same position of a DNA or RNA molecule, then the oligonucleotide andthe DNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotide units which can hydrogen bond witheach other. Thus, "specifically hybridizable" and "complementary" areterms which are used to indicate a sufficient degree of complementaritysuch that stable and specific binding occurs between the oligonucleotideand the DNA or RNA target. It is understood that an oligonucleotide neednot be 100% complementary to its target DNA sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target DNA or RNA moleculeinterferes with the normal function of the target DNA or RNA, and thereis a sufficient degree of complementarity to avoid non-specific bindingof the oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e. under physiological conditions in thecase of in vivo assays or therapeutic treatment, or in the case of invitro assays, under conditions in which the assays are performed.

In particular, oligonucleotides of the invention may be used intherapeutics, as diagnostics, and for research as is specified in thefollowing United States patent applications assigned to the assignee ofthis invention These applications are entitled: Compositions and Methodsfor Modulating RNA Activity, Ser. No. 463,358, filed Jan. 11, 1990;Antisense Oligonucleotide Inhibitors of Papilloma Virus, Ser. No.445,196 Filed Dec. 4, 1989; Oligonucleotide Therapies for Modulating theEffects of Herpesvirus, Ser. No. 485,297, Filed Feb. 26, 1990; Reagentsand Methods for Modulating Gene Expression Through RNA Mimicry Ser. No.497,090, Filed Mar. 21, 1990; Oligonucleotide Modulation of LipidMetabolism, Ser. No. 516,969, Filed Apr. 30, 1990; Oligonucleotides forModulating the Effects of Cytomegalovirus Infections, Ser. No. 568,366,Filed Aug. 16, 1990; Antisense Inhibitors of the Human ImmunodeficiencyVirus, Ser. No. 521,907, Filed May 11, 1990; Nuclease ResistantPyrimidine Modified Oligonucleotides for Modulation of Gene Expression,Ser. No.558,806, Filed Jul. 27, 1990; Novel Polyamine ConjugatedOligonucleotides, Ser. No. 558,663, Filed Jul. 27, 1990; Modulation ofGene Expression Through Interference with RNA Secondary Structure, Ser.No. 518,929, Filed May 4, 1990; Oligonucleotide Modulation of CellAdhesion, Ser. No. 567,286, Filed Aug. 14, 1990; Inhibition of InfluenzaViruses, Ser. No. 567,287, Filed Aug. 14, 1990; Inhibition of Candida,Ser. No. 568,672, Filed Aug. 16, 1990; and Antisense OligonucleotideInhibitors of Papillomavirus, Ser. No. PCT/US90/07067, Filed Dec. 3,1990. These applications disclose a number of means whereby improvedmodulation of RNA and DNA activity may be accomplished througholigonucleotide interaction. In that the specific sequences disclosedtherein may be used in conjunction with the present invention, thedisclosures of the foregoing United States patent applications areincorporated herein by reference.

The following examples are illustrative and are not meant to be limitingof the present invention.

EXAMPLE 1

Isolation of all Sp or all Rp 5'-O-(1-Thiotriphosphate) Nucleoside

5'-O-(1-thiotriphosphate) deoxynucleosides and ribonucleosides areisolated using C-18 reverse phase high performance liquid chromatography(HPLC) using columns packed with ODS Hypersil (Shandon Southern, Runcon,UK) and eluted with an isocratic mixture of solvent A (30 mM potassiumphosphate containing 5 mM tetrabutylammonium ion, pH 7.0) and solvent B(5 mM tetrabutylammonuium hydroxide in methanol). Alternatively,effective separation is achieved using 100 mM triethylammoniumbicarbonate, pH 7.5, containing a linear gradient of acetonitrile from0% to 15% over 20 minutes.

To establish the purity of such HPLC separated enantiomers the HPLCseparated Sp and Rp deoxynucleotide enantiomers are compared tocommercially available deoxynucleoside 5'-O-(1-thiotriphosphates)available from E.I. Dupont, Wilmington, Del.

EXAMPLE 2

Synthesis of Phosphorothioate Extension having Substantially all RpIntersugar Linkages of a Racemic Phosphorothioate Oligonucleotide

Enzymatic synthesis of an all Rp phosphorothioate extension of a racemicphosphorothioate oligonucleotide primer is effected using the modifiedT7 DNA polymerase I, Sequenase™ (U.S. Biochemicals Corp, Cleveland,Ohio). This T7 DNA polymerase is used to extend an 18 merphosphorothioate oligonucleotide primer hybridized to a 21-mer naturalphosphodiester oligonucleotide. 30 picomoles (pmol) of primer andtemplate in a 1× Sequenase™ reaction buffer (U.S. Biochemicals Corp.,Cleveland, Ohio) (final vol 10 μL) are heated for 5 minutes at 95° C.and slowly cooled to room temperature. 180 pmol of deoxy 5'-[α-³⁵S]cytidine triphosphate and Sequenase™ enzyme (U.S. Biochemicals Corp.,Cleveland, Ohio) are added and incubated at 37° C. for 20 minutes. Theproduct is analyzed via polyacrylamide gel electrophoresis (PAGE) usinga 20% polyacrylamide/7M urea denaturing gel. The autoradiograph of theproduct is compared to a control reaction absent primer/template. Thefinal product is subjected to further characterization by, for example,enzymatic degradation. One such degradation is snake venom phosphatasedegradation. A snake venom phosphatase degradation of dinucleosidemonophosphorothioate synthesized using E. coli DNA polymerase I showsthe dinucleoside to be of the Rp configuration.

EXAMPLE 3

Synthesis of Phosphorothioate CGACTATGCAAGTAC (SEQ ID NO:13)Oligonucleotide having Substantially Pure Rp Intersugar Linkages

A large scale enzymatic synthesis of sequence specific all Rpphosphorothioate oligonucleotide was effected utilizing a 55-mer naturalphosphodiester template and a 41-mer natural phosphodiester primer. Thetemplate sequence wasGTACTTGCATAGTCGATCGGAAAATAGGGTTCTCATCTCCCGGGATTTGGTTGAG (SEQ ID NO:14).The primer sequence was CTCAACCAAATCCCGGGAGATGAGAACCCTATTTTCCGATC (SEQID NO:15). The template was selected to have a sequence complementary toa desired specific CGACTATGCAAGTAC (SEQ ID NO:13) sequence. A Sequenase™buffer (U.S. Biochemicals Corp., Cleveland, Ohio) diluted from 5× to 1×was used. The template and primer, both at concentrations of 20 nM areadded to 40 μL of this buffer. The template and primer were hybridizedat 95° C. for 5 minutes and cooled to room temperature. After coolingthe buffer was adjusted to 7 mM DTT. 20 μL of 1:8 diluted Sequenase™enzyme and 320 μM each of Sp GTPαS, CTPαS, ATPαS and TTPαS were thenadded. The reaction solution was adjusted to 140 μL with H₂ O. It wasincubated at 37° C. for 18 hours. The reaction solution was extracted 2×with a like volume of phenol in a standard manner and precipated in astandard manner by treatment with 2.5 volumes of 100% ethanol at -20°C., peltized, washed with 500 μL of 70% ethanol, peltized again anddried. The precipitate was suspended in 20 μL H₂ O for 30 minutes thenadjusted to 1 mM CaCl₂, 25 mM Tris HCl pH 8.0 in 40 μL H₂ O. Thesolution was maintained at 95° C. for 5 minutes and snap cooled, i.e.very quickly cooled with ice. The template and primer were removed fromthe synthesized oligonucleotide by the addition of 4.6 μM DNase I andincubation at 37° C. for 10 minutes. The reaction mixture was phenolextracted 2× and precipitated with ethanol as above. The precipate wasresuspended in H₂ O and purified using 20% polyacrylamide/7M urea gelelectrophoresis coupled with SepPak™ chromatography (Millipore, Milford,Mass.).

In an alternate synthesis, Pst 1 restriction nuclease (LifeTechnologies, Inc., Gaithersburg, Md.) was used to cleave theprimer-bound phosphorothioate oligonucleotide at the restriction site.The desired CGACTATGCAAGTAC (SEQ ID NO:13) phosphorothioateoligonucleotide was purified using polyacrylamide/7M urea gelelectrophoresis coupled with SepPak™ chromatography (Millipore, Milford,Mass.). Yields were optimized using enzymatic cascade effected byrepetitive template-primer addition throughout the reaction. The cascadeaugmented synthesis yielded 75 A₂₆₀ units of the CGACTATGCAAGTAC (SEQ IDNO:13) all Rp configuration phosphorothioate oligonucleotide from a 20mL reaction.

EXAMPLE 4

Synthesis of Phosphorothioate Oligonucleotides Having a Racemic Mixtureof Intersugar Linkages Using Automated DNA Synthesis

Oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 380B) using hydrogenphosphonate chemistry in astandard manner [Agrawal et al., Proc. Natl. Acad. Sci. U.S.A., 85:7079(1988)]. After the final coupling step, the phosphorothioate linkagesare generated by oxidizing the bound oligomer with sulfur in carbondisulfide/triethylamine/pyridine. After sulfur oxidation, standarddeblocking procedures with ammonium hydroxide are used to release theoligonucleotides from the support and remove base blocking groups. Thephosphorothioate oligonucleotides are purified by oligonucleotidepurification column (OPC; ABI, Foster City, Calif.) chromatography andHPLC, using a Beckman System Gold HPLC. The HPLC-purifiedoligonucleotides are then precipitated with ethanol and assessed forfinal purity by gel electrophoresis on 20% acrylamide/7M urea or byanalytical HPLC. The authenticity of the oligonucleotide sequence wasassessed by oxidation with iodine in pyridine/water and standardsequencing methods. These oligonucleotides contain a mixture of allpossible combinations of Rp and Sp isomers at each phosphorous linkage.

EXAMPLE 5

Synthesis of complementary DNA or RNA Sequences Using T7 RNA Polymerasefor Thermodynamic and Kinetic Hybridization Analysis

The synthesis of short complementary DNA oligonucleotides of naturalphosphodiester linkages was performed utilizing standard automatedsynthesis on an ABI model 380B DNA Synthesizer. The oligonucleotides ofcorrect length were purified by HPLC and sequenced by standardtechniques.

T7 RNA polymerase was use for the synthesis of short, complementary RNAoligonucleotides for hybridization analysis. A large amount of T7 RNApolymerase at high concentrations was needed for the many cycles ofinitiation required to synthesize short RNAs. Due to this requirement,the T7 RNA polymerase was derived from a strain of E. coli thatcontained a T7 RNA polymerase expression vector, BL21/pAR1219, obtainedfrom Brookhaven National Laboratory (Upton, N.Y.). The isolation yieldedapproximately 300,000 to 500,000 units of T7 RNA polymerase from 2 L ofcells, absorbance value=1.2 A₆₀₀. This was sufficiently concentrated forsynthesis of short (10-30 nucleotides) RNA species. For synthesis, a T7promoter and a template containing the complementary target sequence andT7 promoter hybridization sequence were synthesized using the ABIsynthesizer (ABI, Foster City, Calif.). Template and promoter werepurified by HPLC to ensure that the correct species was present forenzymatic synthesis. Synthesized products were purified on a 20%polyacrylamide/8M urea gel and sequenced by standard procedures.

EXAMPLE 6

Thermal Denaturation

Oligonucleotides (either phosphorothioate oligonucleotides of theinvention or otherwise) were incubated with either the complementary DNAor RNA oligonucleotides at a standard concentration of 4 μM for eacholigonucleotide in 100 mM ionic strength buffer (89.8 mM NaCl, 10 mMNa-phosphate, pH 7.0, 0.2 mM EDTA). Samples were heated to 90° C. andthe initial absorbance taken using a Guilford Response IIspectrophotometer (Corning). Samples were then slowly cooled to 15° C.and the change in absorbance at 260 nm monitored during the heatdenaturation procedure. The temperature was elevated 1 degree/absorbancereading and the denaturation profile analyzed by taking the firstderivative of the melting curve. Data was also analyzed using atwo-state linear regression analysis to determine the T_(m) and delta G.The results of these tests are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        THERMAL DENATURATION                                                                            SEQ                                                         Sequence          ID NO:   Complement T.sub.m                                 ______________________________________                                        Natural phosphodiester                                                        CGA  CTA    TGC    AAG  TAC   13     DNA      53.2                            CGA  CTA    TGC    AAG  TAC   13     RNA      46.2                            Phosphorothioate with                                                         racemic intersugar linkages                                                   CGA  CTA    TGC    AAG  TAC   13     DNA      46.0                            CGA  CTA    TGC    AAG  TAC   13     RNA      36.5                            Phosphorothioate with                                                         chirally pure intersugar                                                      linkages                                                                      CGA  CTA    TGC    AAG  TAC   13     DNA      45.5                            CGA  CTA    TGC    AAG  TAC   13     RNA      41.5                             GA  CTA    TGC    AAG  TAC   16     DNA      44.5                             GA  CTA    TGC    AAG  TAC   16     RNA      40.0                            ______________________________________                                    

EXAMPLE 7

Synthesis of Radiolabeled Oligonucleotides

Filter binding assays are utilized to quantitate the bindingstringencies of various phosphorothioate oligonucleotides, i.e. theirtendencies to hybridize and form heteroduplexes with DNA or RNA. Theseassays require radiolabeled oligonucleotides.

Phosphorothioate oligonucleotides having all Rp intersugar linkages aresynthesized by enzymatic methods from [³⁵ S]-monomers that have beenpurified from Sp monomers. For automated synthesis of phosphorothioateoligonucleotides containing mixed chirality intersugar linkages,oligonucleotides are synthesized containing hydrogen phosphonates andthen sulfurized in the presence of elemental [³⁵ S] in a pyridine/carbondisulfide mixture. The resulting radiolabeled phosphorothioateoligonucleotide can be purified by OPC chromatography and HPLC. TargetmRNA are applied to nitrocellulose filters and baked at 80° C. for 2hours, blocked and then hybridized with the radiolabeledphosphorothioate oligonucleotide. Binding stringency is assessed byquantitating radiolabeled oligonucleotide eluted from the filters afterincreases in temperature or increases in the ionic strength of aneluting buffer, as for instance, Tris NaCl buffer. Elutedoligonucleotides are also assessed for their mobility in an anionexchange HPLC protocol isocratically utilizing phosphate buffer. Resultsare compared to the mobility of standard oligonucleotides preparedhaving racemic mixtures of intersugar linkages.

EXAMPLE 8

Nuclease Digestion

Determination of the rate of nuclease degradation of thephosphorothioate oligonucleotides in media containing 10% fetal calfserum (FCS) was carried out in Dulbecco's Modified Essential Medium(DMEM) containing 10% heat inactivated FCS. Heat inactivation of the FCSwas carried out at 55° C. for 1 hour prior to addition to media.Oligonucleotides having racemic and chirally pure intersugar linkageswere separately tested for resistance to nuclease digestion. 66 μg/mL ofeach oligonucleotide were separately added to medium and incubated at37° C., at the time intervals indicated in Table 2. 15 μL Aliquots wereremoved and added to 15 μL of 9M urea in 0.1M Tris-HCl (pH 8.3), 0.1Mboric acid and 2 mM EDTA. Aliquots were mixed by vortex and stored at-20° C. Polyacrylamide gel electrophoresis (PAGE) analysis was on 20%polyacrylamide/7M urea slab gels. Following electrophoresis, gels werestained using "Stains All" (Sigma Chem. Co., St. Louis, Mo.). Followingde-staining, gels were analyzed via laser densitometry using anUltraScan XL device (Pharmacia LKB Biotechnology, Uppsala, Sweden).Integrations were performed and the data presented as the percentagedecrease from full length (n) prior to incubation to n-1. These resultsare shown in Table 2 for the oligonucleotide sequence CGACTATGCAAGTAC(SEQ ID NO:6) having Rp chirally pure intersugar linkages.

                  TABLE 2                                                         ______________________________________                                        NUCLEASE DIGESTION                                                            Incubation in 10% fetal calf serum                                            Digestion of oligonuleotide of length n to length n-1                                  Phosphorothioate with                                                                         Phosphorothioate with                                         with racemic intersugar                                                                       chirally pure                                        Time (hours)                                                                           linkages        intersugar linkages                                  ______________________________________                                        0         0                 0                                                 1        44                10                                                 2        45                10                                                 4        54                12                                                 24       70                44                                                 48       70                62                                                 ______________________________________                                    

As is evident from Table 2, the phosphorothioate oligonucleotide havingsubstantially chirally pure intersugar linkages showed greaterresistance to nuclease degradation than did the phosphorothioateoligonucleotide having racemic intersugar linkages.

EXAMPLE 9

RNase H Analysis

Phosphorothioate oligonucleotides having racemic and substantiallychirally pure intersugar linkages were analyzed for susceptibility toRNase H. Oligonucleotides (2-fold molar excess to RNA) and 5 μg (3.1 kb)in vitro synthesized mRNA (using T7 RNA polymerase promoter) wereincubated in 5 μL RNase H hybridization buffer for 30 minutes at 60° C.Samples were slowly cooled to room temperature and then adjusted to 3.7mg/mL BSA, 20 units E. coli RNase H (Promega), 142 mM DTT, 150 mM KCl,and 3 mM MgCl₂. Samples were incubated for 30 minutes at 37° C. Sampleswere then extracted with phenol, precipitated with ethanol, and analyzedby electrophoresis on 1.2% agarose gels following ethidium bromidestaining. Markers were run on gels concurrently with the samples todetermine approximate length of RNA samples.

EXAMPLE 10

A patient suffering from hepatitis caused by HCV is treated with Oligo#259 (SEQ ID NO:1), Oligo #260 (SEQ ID NO:2), Oligo #270 (SEQ ID NO:3),Oligo #330 (SEQ ID NO:4) or Oligo #340 (SEQ ID NO:5), each of which aresynthesized according to the procedure of Example 3 or Example 19.1-1000 μg/kg body weight of oligonucleotide is incorporated into apharmaceutically acceptable carrier and administered intravenously orintramuscularly. Treatment may be repeated as necessary until thedisease has been ablated.

EXAMPLE 11

A patient suffering from an inflammatory disease mediated by ICAM-1 istreated with ISIS-2302, an oligonucleotide synthesized according toExample 3 or Example 19, and having the sequence GCCCAAGCTGGCATCCGTCA(SEQ ID NO:6). 1-1000 μg/kg body weight of oligonucleotide isincorporated into a pharmaceutically acceptable carrier and administeredintravenously or intramuscularly. Treatment may be repeated as necessaryuntil the disease has been ablated.

EXAMPLE 12

A patient suffering from retinitis caused by cytomegalovirus is treatedwith ISIS-2922, an oligonucleotide synthesized according to Example 3 orExample 19, and having the sequence GCGTTTGCTCTTCTTCTTGCG (SEQ ID NO:7).1-1000 μg/kg body weight of oligonucleotide is incorporated into apharmaceutically acceptable carrier and administered intravitreally.Treatment may be repeated as necessary until the infection is ablated.

EXAMPLE 13

A patient suffering from PKC-α-mediated cancer is treated withISIS-3521, an oligonucleotide synthesized according to Example 3 orExample 19, and having the sequence GTTCTCGCTGGTGAGTTTCA (SEQ ID NO:8).1-1000 μg/kg body weight of oligonucleotide is incorporated into apharmaceutically acceptable carrier and administered intravenously orintramuscularly. Treatment may be repeated as necessary until thedisease has been ablated.

EXAMPLE 14

A patient suffering from C-raf kinase-mediated cancer is treated withISIS-5132, an oligonucleotide synthesized according to Example 3 orExample 19, and having the sequence TCCCGCCTGTGACATGCATT (SEQ ID NO:9).1-1000 μg/kg body weight of oligonucleotide is incorporated into apharmaceutically acceptable carrier and administered intravenously orintramuscularly. Treatment may be repeated as necessary until thedisease has been ablated.

EXAMPLE 15

A patient suffering from C-raf kinase-mediated cancer is treated withISIS-2503 (SEQ ID NO:10), ISIS-2570 (SEQ ID NO:11) or ISIS-6957 (SEQ IDNO:12), each of which are synthesized according to the procedure ofExample 3 or Example 19. 1-1000 μg/kg body weight of oligonucleotide isincorporated into a pharmaceutically acceptable carrier and administeredintravenously or intramuscularly. Treatment may be repeated as necessaryuntil the disease has been ablated. Compounds 1, 2 and 3 of Examples 16,17 and 18, respectively, are synthesized according to the procedure ofStec et al. [Nucleic Acids Res., 19:5883 (1991)] and Stec andLesnikowski [Methods in Molecular Biology, S. Agrawal, Ed., Volume 20,p. 285, 1993].

EXAMPLE 16

Synthesis of 2-Chloro-1,3,2-Oxathiaphospholane (1)

A mixture of pyridine (1 mol), benzene (400 mL), 2-mercaptoethanol (0.5mol) and phosphorus trichloride (0.5 mol) are stirred at roomtemperature for 30 minutes. Pyridinium chloride is filtered off, solventis evaporated under reduced pressure and crude product is purified bydistillation under reduced pressure. The fraction boiling at 70°-72°C./20 mm Hg is collected and characterized by ³¹ P NMR.

EXAMPLE 17

Synthesis of N,N-Diisopropylamino-1,3,2-Oxathiaphospholane (2)

Compound 1 (0.2 mol) is dissolved in n-pentane (300 mL) anddiisopropylamine (0.4 mol) is added dropwise. The reaction mixture isstirred at room temperature for 30 minutes, after which diisopropylaminehydrochloride is filtered off, solvent is evaporated under reducedpressure and crude product is purified by vacuum distillation. Product 2is obtained as the fraction boiling at 70° C./0.1 mm Hg and ischaracterized by ³¹ P NMR and mass spectroscopy.

EXAMPLE 18

Synthesis of5'-O-Dimethoxytritylthymidine-3'-O[2-Thiono-1,3,2-Oxathiaphopsholane](3)

5'-O-Dimthoxytritylthymidine (10 mmol) and 1H-tetrazole (11 mmol) arevacuum dried and dissolved in dichloromethane (25 mL). Compound 2 (11mmol) is added to the solution and the reaction mixture is stirred atroom temperature for 2 hours. Dried elemental sulfur (15 mmol) is addedand the reaction mixture is stirred and left at room temperature for 16hours. Unreacted sulfur is then filtered off and the filtrate isconcentrated under reduced pressure. The residue is dissolved inchloroform (3 mL) and purified by silica gel (230-400 mesh) columnchromatography, eluting first with chloroform, and next withchloroform:methanol (97:3). Individual diastereomeric species ofcompound 3 are obtained by column chromatography on silica gel. Compound3 is dissolved in ethyl acetate and applied on a silica gel 60H column.Ethyl acetate is used as the eluting solvent, and elution is monitoredby HPTLC (silica gel 60, ethyl acetate as the developing solvent).Fractions containing separated diastereomers of compound 3 areconcentrated under reduced pressure and the residue is characterized by³¹ P NMR and HPLC (Lichrospher Si100, 5 μM, ethyl acetate as eluant,flow rate 3 mL/minute). The fast eluting fraction corresponds to the Spdiastereomer, and the slow eluting fraction is the Rp diastereomer.

EXAMPLE 19

Stereospecific Control of Reaction Between 5'-OH Nucleosides andDiastereomerically Pure 3 in Solid Phase Automated Synthesis

The procedure of Stec et al. was followed using an Applied Biosystems(Foster City, Calif.) model 380B automated DNA synthesizer. The reactionbetween a 5'-OH nucleoside and diastereomerically pure nucleosideoxathiaphospholane, such as 3, requires the use of1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) as the catalyst. Because thecommercially available linker used for the attachment of oligonucleotideto support matrix in automated DNA synthesis is unstable to DBU, amodification is required in the linker. A suitable linker foroligonucleotide synthesis via the oxathiaphospholane method is a"succinic-sarcosinyl" linker that is resistant to DBU, and can behydrolyzed by concentrated ammonium hydroxide at room temperature inless than 1 hour.

(A) Synthesis of 5'-O-dimethoxytritylnucleosides bound to solid matrixvia "succinic-sarcosinyl" linker:

(1) N-Fmoc-sarcosine (Bachem Bioscience, Inc., Philadelphia, Pa.) (1.6mmol) is added to long chain alkyl amine-CPG (LCA-CPG, Sigma, St. Louis,Mo.) (2 g) and dried under vacuum. Anhydrous DMF (5 mL), pyridine (0.5mL) and DCC (2.4 mmol) are added and the reaction mixture is shaken atroom temperature for 12 hours. The solvent is then filtered off and thesupport is washed with methanol:acetonitrile:pyridine (1:1:1, 3×20 mL).The N-Fmoc protecting group is removed by treating the support with 10mL of a 10% solution of piperidine in pyridine. N-sarcosinylated LCA-CPGis washed with methanol:acetonitrile:pyridine (1:1:1, 3×20 mL) and driedunder vacuum.

(2) 5'-O-Dimethoxytritylnucleoside is added to the sarcosinylatedLCA-CPG obtained as described in (1) in the presence of DMF (2 mL),pyridine (0.2 mL) and DCC (50 mg). The reaction mixture is shaken atroom temperature for 12 hours and then washed withmethanol:acetonitrile:pyridine (1:1:1, 3×20 mL). After drying, thesupport is treated with N-methylimidazole:THF (1 mL) and aceticanhydride/lutidine (1 mL) for 15 minutes. The support is then washedwith methanol:acetonitrile:pyridine (1:1:1, 3×10 mL), followed byacetonitrile (3×10 mL), and then dried under vacuum.

(B) Diastereomerically pure activated nucleosides are subsequently addedonto the oligonucleotide attached to the sarcosinyl LCA-CPG support inthe presence of a 300-fold molar excess of DBU. The diastereomers ofactivated nucleosides are separated by column chromatography [silica gel60H, ethyl acetate is used as the eluting solvent, elution is monitoredby HPTLC (silica gel 60, ethyl acetate as the developing solvent)] priorto use in the coupling reaction. The synthetic protocol is shown inTable 3.

                  TABLE 3                                                         ______________________________________                                        Chemical steps for one synthesis cycle                                                                     Time                                             Reagent or solvent                                                                              Purpose    (minutes)                                        ______________________________________                                        Trichloroacetic acid in                                                                         Detritylation                                                                            1.5                                              dichloromethane (2:98)                                                        Acetonitrile      Wash       2                                                Activated nucleoside (with                                                                      Coupling   10                                               DBU) in acetonitrile                                                          Acetonitrile      Wash       2                                                Acetic anhydride/lutidine                                                                       Capping    1                                                in THF (1:1:8) and                                                            N-methylimidazole in THF                                                      (4:21)                                                                        Acetonitrile      Wash       1                                                ______________________________________                                    

The diastereomeric purity of the phosphorothioate oligonucleotide can bedetermined by ³¹ P NMR, by HPLC (Lichrospher Si100, 5 μM, ethyl acetateas eluant, flow rate 3 mL/minute), enzymatically or by electrophoreticmethods.

EXAMPLE 20

Treatment of a Disease State in a Human Patient

The oligonucleotides of the invention may be used for treatment ofvarious disease states. Treatment of a patient diagnosed with aparticular disease state comprises administration of an effective doseof the olignucleotide, in a pharmaceutically accepted formulation, tothe patient via an appropriate route. The effective oligonucleotide dosedepends on the disease state being treated, the severity of the diseasestate and the age of the patient being treated. The effective dose of anoligonucleotide may be determined based on its IC₅₀ and is a routineprocedure for one of skill in the art. Alternatively, the effective doseof the oligomer may be determined by using the pharmacokinetics softwareprogram TopFit. For example, dosage of oligonucleotides may vary from0.01 μg (for children) to 100 g (for adults) per kg of body weightdepending on progression of the disease state. Similarly, the frequencyof dosing depends on the progression of the disease state and may varyfrom once or more daily to once every 20 years.

The route of oligonucleotide administration depends on the disease statebeing treated. For example, administration of an oligonucleotide to apatient being treated for an inflammatory disorder may be accomplishedeither via oral or rectal routes. For treatment of a patient afflictedwith AIDS, the most effective method of oligonucleotide administrationmay be an oral route or by subcutaneous injection. Cancers such asbreast cancer may be treated via subcutaneous injection, while coloncancer may be treated via oral or rectal administration of theoligonucleotide. Diseases or disorders of the central nervous system maybest be treated by intrathecal or intraventricular administration fordelivery of the oligonucleotide to the spinal column or the brain of thepatient.

Following oligonucleotide administration, the patient may be monitoredfor alleviation of symptoms associated with the disease state.Subsequently, the dosage may be adjusted (increased or decreased)depending upon the severity and amenability of the disease state totreatment.

It may be preferable to administer oligonucleotides of the invention incombination with other traditional therapeutics. The oligonucleotidesmay be administered in combination with drugs including, but not limitedto, AZT for the treatment of patients afflicted with AIDS, sulfasalazinefor the treatment of an inflammatory disorder such as ulcerativecolitis, and 5-fluorouracil for the treatment of colon cancer.

Also, it may be desirable to administer maintenance therapy to a patientwho has been successfully treated for a disease state. The dosage andfrequency of oligonucleotide administration as part of a maintenanceregimen may vary from 0.01 μg to 100 g per kg of body weight, rangingfrom once or more daily to once every several years.

EXAMPLE 21

Intraventricular Administration of Oligonucleotides

Intraventricular drug administration, for the direct delivery of drug tothe brain of a patient, may be desired for the treatment of patientswith diseases afflicting the brain. To effect this mode ofoligonucleotide administration, a silicon catheter is surgicallyintroduced into a ventricle of the brain of a human patient, and isconnected to a subcutaneous infusion pump (Medtronic Inc., Minneapolis,Minn.) that has been surgically implanted in the abdominal region[Cancer Research, 44:1698 (1984)]. The pump is used to inject theoligonucleotides and allows precise dosage adjustments and variation indosage schedules with the aid of an external programming device. Thereservoir capacity of the pump is 18-20 mL and infusion rates may rangefrom 0.1 mL/h to 1 mL/h. Depending on the frequency of administration,ranging from daily to monthly, and the dose of drug to be administered,ranging from 0.01 μg to 100 g per kg of body weight, the pump reservoirmay be refilled at 3-10 week intervals. Refilling of the pump isaccomplished by percutaneous puncture of the self-sealing septum of thepump.

EXAMPLE 22

Intrathecal Administration of Oligonucleotides

Intrathecal drug administration for the introduction of drug into thespinal column of a patient may be desired for the treatment of patientswith diseases of the central nervous system. To effect this route ofoligonucleotide administration, a silicon catheter is surgicallyimplanted into the L3-4 lumbar spinal interspace of a human patient, andis connected to a subcutaneous infusion pump which has been surgicallyimplanted in the upper abdominal region [The Annals of Pharmacotherapy,27:912 (1993) and Cancer, 41:1270 (1993]. The pump is used to inject theoligonucleotides and allows precise dosage adjustments and variations indose schedules with the aid of an external programming device. Thereservoir capacity of the pump is 18-20 mL, and infusion rates may varyfrom 0.1 mL/h to 1 mL/h. Depending on the frequency of drugadministration, ranging from daily to monthly, and dosage of drug to beadministered, ranging from 0.01 μg to 100 g per kg of body weight, thepump reservoir may be refilled at 3-10 week intervals. Refilling of thepump is accomplished by a single percutaneous puncture to theself-sealing septum of the pump.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 16                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCTTTCGCGACCCAACACTA20                                                        (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GCCTTTCGCGACCCAACACT20                                                        (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GTACCACAAGGCCTTTCGCG20                                                        (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECLYLE TYPE: DNA (genomic)                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GTGCTCATGGTGCACGGTCT20                                                        (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TTTAGGATTCGTGCTCATGG20                                                        (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCCCAAGCTGGCATCCGTCA20                                                        (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GCGTTTGCTCTTCTTCTTGCG21                                                       (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GTTCTCGCTGGTGAGTTTCA20                                                        (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRAMEDNESS: single                                                       (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TCCCGCCTGTGACATGCATT20                                                        (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TCCGTCATCGCTCCTCAGGG20                                                        (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CCACACCGACGGCGCCC17                                                           (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CAGTGCCTGCGCCGCGCTCG20                                                        (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CGACTATGCAAGTAC15                                                             (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GTACTTGCATAGTCGATCGGAAAATAGGGTTCTCATCTCCCGGGATTTGG50                          TTGAG55                                                                       (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO;15:                                      CTCAACCAAATCCCGGGAGATGAGAACCCTATTTTCCGATC41                                   (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CKMCTERISTICS:                                                   (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GACTATGCAAGTAC14                                                              __________________________________________________________________________

What is claimed is:
 1. An oligonucleotide represented by SEQ ID NO:6wherein at least 75% of the nucleoside units are joined together by Spphosphorothioate 3' to 5' linkages.
 2. An oligonucleotide represented bySEQ ID NO:6 wherein at least 75% of the nucleoside units are joinedtogether by Rp phosphorothioate 3' to 5' linkages.
 3. Theoligonucleotide of claim 1 wherein all of the nucleoside units arejoined together by Sp phosphorothioate 3' to 5' linkages.
 4. Theoligonucleotide of claim 2 wherein all of the nucleoside units arejoined together by Rp phosphorothioate 3' to 5' linkages.
 5. Acomposition containing an oligonucleotide of claim 1 and an acceptablecarrier.
 6. A composition containing an oligonucleotide of claim 2 andan acceptable carrier.